Increased calreticulin stability in differentiated NG-108-15 cells correlates with resistance to apoptosis induced by antisense treatment.
Since its first identification as a high-affinity calcium-binding protein over two decades ago [T.J. Ostwald and D.H. MacLennan, Isolation of a high-affinity calcium-binding protein from sarcoplasmic reticulum, J. Biol. Chem., 249 (1974) 974-979], calreticulin has become recognized as a multifunctional protein involved in a wide variety of cellular processes. We have previously shown that it has a protective function in Ca2+-mediated cell death [N. Liu, R.E. Fine, E. Simons and R.J. Johnson, Decreasing calreticulin expression lowers the Ca2+ response to bradykinin and increases sensitivity to ionomycin in NG-108-15 cells, J. Biol. Chem. , 269 (1994) 28635-28639]. We report here that in NG-108-15 neuroblastomaxglioma hybrid cells, calreticulin protein levels increase markedly when these cells are induced to differentiate by treating them with N,N-dibutyryl cAMP (db-cAMP). We demonstrate that the reason for this increase is mostly due to a large increase in the turnover time of calreticulin in differentiated cells. We also show that a calreticulin antisense oligonucleotide, CrtAS1, previously described by Liu and co-workers [N. Liu, R.E. Fine, E. Simons and R.J. Johnson, Decreasing calreticulin expression lowers the Ca2+ response to bradykinin and increases sensitivity to ionomycin in NG-108-15 cells, J. Biol. Chem., 269 (1994) 28635-28639] causes cell death in undifferentiated NG-108-15 cells when antisense treatment is extended for more than 24 h. This effect is not seen in NG-108-15 cells that have been induced to differentiate with db-cAMP until the cells have been treated with antisense for more than 4 days, due to the increased stability of Crt in these cells. Our results indicate that the mechanism by which these cells die is likely to be apoptosis.
Johnson
R J
RJ
Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA. rjohnson@bu.edu
Liu
N
N
Shanmugaratnam
J
J
Fine
R E
RE
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001483:Base Sequence; D003994:Bucladesine / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D037282:Calreticulin; D002454:Cell Differentiation; D005910:Glioma; D006822:Hybrid Cells; D018832:Molecular Chaperones / Q000378:metabolism; D009447:Neuroblastoma; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012261:Ribonucleoproteins / Q000096:biosynthesis / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D018832:Molecular Chaperones; D016376:Oligonucleotides, Antisense; D012261:Ribonucleoproteins; D003994:Bucladesine